Plant biomass or lignocellulose is the most plentiful renewable energy source on earth, and holds great promise as an alternative energy source to oil. The main component of plant biomass dry weight is lignocellulose, which is composed of polysaccharides such as cellulose and hemicellulose, and lignin. For example, polysaccharides can be hydrolyzed by a glycoside hydrolase such as a cellulase or hemicellulase to form monosaccharides such as glucose and xylose, which can then be used as the raw materials for biofuels or chemical products.
Lignocellulose is recalcitrant due to its complex structure, and is difficult to degrade or hydrolyze with a single glycoside hydrolase. The complete hydrolysis of lignocellulose generally requires three types of enzymes, namely an endoglucanase (cellulase or endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21), and it is thought that the addition of a further plurality of enzymes including the hemicellulase xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and other plant cell wall-degrading enzymes such as β-xylosidase (EC 3.2.1.37) is also necessary.
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the hydrolyzed biomass solution is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis process at a high temperature, for example 75° C. or higher, the rate of the hydrolysis reaction can be increased, and the viscosity of the hydrolyzed biomass solution can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermal stability is very desirable.
Cellobiohydrolases play the most important role in the lignocellulose hydrolysis process, but the majority of these cellobiohydrolases are derived from filamentous fungi, and generally have quite poor thermostability. Only a handful of thermostable cellobiohydrolases having an optimum temperature exceeding 65° C. have been reported (for example, see Patent Document 1). A large number of thermostable endoglucanases have already been isolated from thermophilic bacteria and filamentous fungi for purposes such as lignocellulose degradation, treatment agents for cellulose fibers and paper pulp processes (for example, see Patent Document 2). In terms of thermostable cellulase multienzymes having linked catalytic domains, CelA from the thermophilic bacterium Caldicellulosiruptor bescii has been reported, and it has been reported that compared with the case where the catalytic domains of GH family (Glycoside Hydrolase family) 9 and GH family 48 are used individually, the cellulose degradation activity can be improved when these catalytic domains are linked (see Non-Patent Document 1).
However, there are currently no reported examples of naturally derived enzymes in which a thermostable cellobiohydrolase and a thermostable endoglucanase are linked. An enzyme prepared by artificially linking a cellobiohydrolase from the filamentous fungus Trichoderma reesei and an endoglucanase from the theromphilic bacterium Acidothermus cellulolyticus has been reported, but in addition to the linked enzyme protein, cleaved endoglucanase fragments are also produced, and the process is inefficient (see Patent Document 3, Non-Patent Document 2).